Dementia is a condition of deteriorating mentality that is characterized by marked decline in the individual""s former intellectual level, including memory loss, impaired judgment, speech and orientation, and is often accompanied by emotional apathy. (WEBSTER""S MEDICAL DESK DICTIONARY, Merriam-Webster, Inc., Springfield, Mass., p.169 (1986)).
A leading cause of dementia is Alzheimer""s disease,(AD), a neurodegenerative disorder affecting 17 to 20 million people worldwide (Yamazaki, T., et al., J. Cell. Biol., 129;431-442 (1995); Brinaga, M., Science, 269:917-918 (1995); Lavy-Lahad, E., et al., Science, 269:970-972 (1995); Lavy-Lahad, E., et al, Science, 269:973-977 (1995)). AD is characterized by progressive dementia together with neuropathological findings of xe2x80x9csenile plaquesxe2x80x9d in the brain formed by deposits of xcex2-amyloid protein, surrounded by clusters of degenerating neurons. xcex2-amyloid protein itself is a fragment of the 770 amino acid membrane bound xcex2-amyloid precursor protein (xcex2APP) that is expressed in both neuronal and non-neuronal tissues.
Muteins of xcex2APP have been produced for the purpose of developing a xcex2APP substrate system wherein xcex2APP is cleavable or not cleavable such that xcex2AP producing enzymes and inhibitors thereof may be isolated.
The specific cause of Alzheimer""s disease has not yet been determined. A mutation in the xcex2APP gene in families with one form of autosomal dominant AD was found to be associated with increased xcex2-amyloid synthesis and aggregation in the brain. A receptor for xcex2APP has been identified as the low density lipoprotein receptor-related protein, ApoE, and it has been postulated that this receptor protein, the enzyme responsible for xcex2APP cleavage in the cell membrane, production of xcex2APP and/or production of extracellular matrix molecules may be abnormal individually or in combination in AD patients, resulting in excess xcex2-amyloid deposition and the observed neurotoxicity. However, the mechanism by which other known xcex2APP gene mutations cause AD, as well as the pathophysiology of non-familial AD in which xcex2APP gene mutations have not been recognized, is not understood.
Therefore, diagnosing Alzheimer""s disease as the cause of an individual""s dementia, as well as treating AD and developing drug therapies is very difficult. Although recent reports of using Positron-emission tomography (PET) (Reiman, E. M., et al., New Eng. J Med., 334:752-758 (1996), determining the genotype of an individual""s ApoE, or measuring the levels of xcex2-amyloid protein in cerebral spinal fluid may be promising, diagnosis of Alzheimer""s is currently confirmed only upon autopsy to determine the presence of xcex2-amyloid senile plaques.
In vitro systems employed to study Alzheimer""s disease to date consist of malignant, or transformed cells that are not of neural crest origin, or lower vertebrate neuronal cultures. It would be of great advantage to have an Alzheimer""s disease model system using normal human neural crest-derived cells. However, to date, no such model system has been developed.
Moreover, recent studies have shown that damage to CNS neurons due to Alzheimer""s disease begins years before clinical symptoms are evident. (Reiman, E. M., et al., New Eng. J Med., 334:752-758 (1996)), suggesting that therapy could begin in the pre-symptomatic phase of the disease if a sensitive diagnostic test and targeted therapies were available. There exists a great need to determine the physiological mechanisms involved with the disease and for an accurate and easy to perform assay to evaluate the risk of developing Alzheimer""s disease.
The present invention relates to the discovery that neurons and epidermal melanocytes are neural crest-derived cells that undergo xcex2-amyloid mediated apoptosis mediated by xcex2-amyloid binding to the same receptor, the 75 kD neurotrophin receptor (p75NTR). As used herein, the term xcex2-amyloid protein is intended to encompass xcex2-amyloid protein (a 4.2 kD polypeptide (Selkoe, D. J., Neuron, 6:487-498 (1991); Glenner G. G. and Wong, C. W., Biochem. Biophys. Res. Commun., 120:885-890 (1993), the teachings of which are herein incorporated by reference), xcex2-amyloid precursor protein (xcex2APP), and fragments of xcex2-amyloid and xcex2-amyloid precursor protein referred to herein as xcex2-amyloid peptides, including xcex2-amyloid 1-40 peptide, xcex2-amyloid 1-42 peptide, xcex2-amyloid 25-36 peptide or xcex2-amyloid 28-30 peptide. (xcex2-amyloid protein is also referred to herein as xcex2-amyloid).
More specifically, it is demonstrated herein that xcex2-amyloid protein or peptide binds to the p75 nerve growth factor receptor (p75NTR) of neural crest-derived cells, e.g. melanocytes, resulting in apoptosis of the cell. It is further demonstrated herein that inhibiting the binding of xcex2-amyloid protein or peptide to the p75 nerve growth factor receptor, results in inhibiting the activation of the p75 nerve growth factor receptor, which in turn inhibits apoptosis.
The present invention relates to methods of inhibiting xcex2-amyloid-mediated activation of the p75 nerve growth factor receptor of a cell that expresses the p75 nerve growth factor receptor, methods of inhibiting the binding of xcex2-amyloid protein and xcex2-amyloid peptides to the p75 nerve growth factor receptor, and methods of inhibiting xcex2-amyloid-mediated apoptosis of neural crest-derived cells. The methods comprise contacting the cell with a substance, comprising, for example, the amino acid sequence lysine-glycine-lysine (KGK) or lysine-glycine-alanine (KGA), wherein the substance binds to the p75 nerve growth factor receptor, resulting in the inhibition of xcex2-amyloid protein or xcex2-amyloid peptide binding to and/or activation of the p75 nerve growth factor receptor, or wherein the substance inhibits xcex2-amyloid protein or xcex2-amyloid peptide mediated apoptosis of the cell which expresses the p75 nerve growth factor receptor.
It is has been reported that cell death receptors mediate apoptosis by aggregation resulting from ligand binding or membrane perturbation. Applicants have demonstrated that xcex2-amyloid aggregates the p75NTR thereby inducing cell death. Peptides, specifically cyclic peptides, bind the receptors individually and block aggregation of the p75NTR, thereby inhibiting apoptosis. Specifically encompassed by the present invention are cyclic peptides (e.g., peptides in a xcex2-loop conformation) which comprise lysine-glycine-lysine or lysine-glycine-alanine, or other sequences capable of binding to the p75NTR. 
Activation of the p75 nerve growth factor receptor can be determined by measuring the xcex2-amyloid activation of the p75 nerve growth factor receptor of neural crest-derived cells, in culture or in a tissue sample, in the presence of the test substance and comparing the results with the xcex2-amyloid activation of the p75 nerve growth factor receptor of neural crest-derived cells in a control culture or sample without the test-substance. A decrease of xcex2-amyloid activation of the p75 nerve growth factor receptor of neural crest-derived cells in the test sample compared to xcex2-amyloid activation of the p75 nerve growth factor receptor of neural crest-derived cells in the control sample is indicative of a substance that inhibits xcex2-amyloid-mediated apoptosis in neural crest-derived cells.
The present invention further relates to in vitro methods of screening substances and identifying those substances capable of inhibiting, or decreasing cell apoptosis mediated by xcex2-amyloid, or activation of the p75NTR, and to substances identified by these methods.
The method of identifying substances that inhibit xcex2-amyloid-mediated apoptosis of cells that express the p75 nerve growth factor receptor comprises contacting the cells, in culture or in a tissue sample, with xcex2-amyloid protein, or peptide and with the substance to be tested, wherein the xcex2-amyloid protein/peptide is at a concentration sufficient to bind to and activate the p75 nerve growth factor receptor, thereby producing a test sample. The test sample is maintained under conditions sufficient for the xcex2-amyloid protein or peptide or test-substance to bind to the p75 nerve growth factor receptor expressed on the cells and activation of the p75 nerve growth factor receptor is determined, thereby identifying substances that inhibit apoptosis.
The present invention further relates to methods of diagnosing and treating Alzheimer""s disease, and other neurodegenerative diseases mediated by xcex2-amyloid protein, or by aberrant activation of the low affinity nerve growth factor receptor localized on neural cell surfaces. For example, autoimmune encephalomyelitis, Huntington""s disease, Pick""s disease, corticobasal degeneration, progressive supra-nuclear palsy, Gerotman-Shausslesr Scheinker syndrome, Niemann-Pick disease, Down""s Syndrome and progressive supranuclear palsy are encompassed by this invention.
Neurodegenerative diseases mediated by xcex2-amyloid protein encompass diseases affecting neural crest-derived cells, such as central nervous system (CNS) neurons and in which xcex2-amyloid protein, PAPP, or xcex2-amyloid peptides initiate, or exacerbate, a process leading to neuron degeneration, or neuronal cell death. Neurodegenerative diseases mediated by aberrant activation of the low affinity nerve growth factor encompasses diseases in which the low affinity nerve growth factor is activated by a substance resulting in apoptotic cell death. The neurodegenerative diseases encompassed by the present invention are characterized by progressive dementia in the affected individual. Specifically encompassed by the present invention is the neurodegenerative disease, Alzheimer""s disease (AD), which is characterized by the deposition of -amyloid peptides in neural tissue, leading to neuronal cell degeneration, cell death and progressive dementia.
The present invention also relates to methods of evaluating the risk of an individual to develop Alzheimer""s disease using an in vitro assay system comprising epidermal melanocytes obtained from the individual. Epidermal melanocytes are melanocytes found in the epidermis (skin) and hair bulbs of vertebrates. The present invention is based on Applicants"" findings that human melanocytes exhibit significant similarities with central nervous system neurons (the cells predominantly affected by Alzheimer""s disease) and that melanocytes utilize the same signaling molecules as neurons to determine their survival versus programmed cell death (apoptosis).
For example, neuronal cells express a high affinity (p140trkA) and a low affinity receptor (p75NTR) for nerve growth factor (NGF). As described herein, Applicants have demonstrated that these nerve growth factor receptors are also expressed on melanocytes and that xcex2-amyloid binds to the low affinity nerve growth factor receptor, p75NTR, expressed on the melanocyte surface. Applicants also demonstrate herein that binding of xcex2-amyloid to the p75NTR activates the receptor, resulting in apoptotic cell death of the melanocytes. Applicants further demonstrate that the xcex2-amyloid mediated-apoptosis can be competitively blocked by providing nerve growth factor or a biologically active fragment, analog or derivative thereof. Nerve growth factor is a physiologic ligand for p75NTR that has a 2-3-fold higher receptor affinity than xcex2-amyloid and when binding coordinately to p75NTR and p140trkA, exhibits a 200-fold higher affinity
Additionally, Applicants have demonstrated that melanocytes secrete xcex2-amyloid (in the form of its precursor protein) constitutively, and in increased amounts in response to trauma, e.g., UV irradiation. Thus, based on results described herein,
Applicants have established that melanocytes, easily obtainable from skin biopsies, are reasonable model cells for the study and diagnosis of Alzheimer""s disease.
Based on the results described herein, neuronal cell death resulting in Alzheimer""s Disease can arise in several different settings. For example, if an excess of xcex2-amyloid protein is present in the central nervous system (CNS), more xcex2-amyloid protein will bind to the p75NTR and initiate the apoptotic pathway in cells. Nerve growth factor binds to two receptors on the cell surface, p75NTR and p140trkA. Nerve growth factor binding to both of these receptors leads to the transmission of a cell survival signal. If excess amounts of the p75NTR are expressed on cells, more xcex2-amyloid can bind to the p75NTR even in the presence of NGF and initiate the cell death pathway. If insufficient p140trkA is expressed, which results in an excess of p75NTR on the cell surface, or if insufficient nerve growth factor ligand is available to bind to the p75NTR, xcex2-amyloid can then also bind to the excess p75NTR, leading to cell death. All of the above, or any combination of the above, can lead to apoptosis and AD.
In the proposed diagnostic test, ease of induction of melanocyte apoptosis in vitro following exposure to xcex2-amyloid protein and/or the blocking of this apoptosis by nerve growth factor (NGF) supplementation is correlated with the predisposition of the cell donor to develop Alzheimer""s disease. Melanocytes obtained from a patient""s skin biopsy are compared to standardized control cell lines. xcex2-amyloid protein or peptide is introduced into the cultures and melanocyte apoptosis resulting from binding of the xcex2-amyloid protein to the p75NTR is determined. Activation of the p75NTR by binding of the p75NTR by xcex2-amyloid protein, or peptide, results in apoptotic cell death of the melanocytes. Thus, activation of the p75NTR by xcex2-amyloid can be measured quantitatively as apoptotic cell death of the melanocytes. Apoptotic cell death is readily assessed by any of a number of standard parameters such as propidium iodine incorporation into nuclear fragments, labeling of DNA strand breaks using fluorescein tagged dUTP in the presence of terminal deoxynucleotidyl transferase (TUNEL reaction), or by demonstration of fragmented DNA (a DNA ladder). Apoptosis can also be measured by cell counts that reveal progressive cell death. The activation of the p75NTR expressed on the melanocytes obtained from the individual suspected of having AD is compared with the activation of the p75NTR expressed on control melanocytes. If the activation of the p75NTR on the individual""s melanocytes is greater than the activation of the p75NTR on the control melanocytes, it is indicative of the greater risk of the individual to develop Alzheimer""s disease.
The present invention also relates to methods of treating, or reducing the risk of developing, Alzheimer""s disease, or other neurodegenerative diseases associated with the xcex2-amyloid protein, or by the activation of the p75NTR resulting in apoptotic neural cell death. In the proposed therapy for Alzheimer""s disease, a composition containing the tripeptide lysine-glycine, lysine, lysine-glycine-alanine or a similar peptide with established affinity for the p75NTR would be delivered by an appropriate route (e.g., intravenously with an agent to reduce the blood-brain barrier or intraventricularly) to central nervous system neurons at risk of xcex2-amyloid induced apoptotic cell death to block xcex2-amyloid binding to p75NTR Nerve growth factor (NGF), biologically active fragments, analogs or derivatives of NGF (wherein biological activity is defined herein as the ability of the fragment, analog or derivative to bind to the p75NTR expressed on neural derived cells such as neurons and melanocytes) and/or other neurotropins could also be administered to further compete against xcex2-amyloid binding to p75NTR, as well as to activate cell survival programs within damaged neurons, for example through upregulation of the apoptosis-inhibiting protein, Bcl-2. The therapeutic peptide is designed to have a receptor affinity comparable to, or greater than, that of xcex2-amyloid but less than that of NGF and other neurotrophins. Typically, the peptide will be a cyclic, or xcex2-loop configuration.
Other methods encompassed by the present invention include methods of decreasing, or inhibiting the expression of p75NTR by contacting cells expressing the receptor wtih an anti p75NTR anti sense nucleic acid, thereby decreasing or inhibiting the expansion of p75NTR on the cells, thus reducing the risk of xcex2-amyloid mediated apoptosis. Anti-sense p75NTR nucleic acid molecules can be produced by one of skill in the art using the nucleotide sequence of p75NTR as described in Johnson, D., et al., Cell,. 47:545-554 (1986).
Alzheimer""s disease is a devastating and ultimately fatal disorder. Early detection of Alzheimer""s disease would make possible early intervention to prevent, or substantially reduce, neuron degeneration and death. Currently, therapies to treat Alzheimer""s disease are minimally effective. As effective treatments become available, it would permit rational therapy from an early point in the disease process when secure diagnosis by conventional clinical criteria is rarely possible. Availability of a therapeutic substance that could slow the progression of Alzheimer""s disease, or other neurodegenerative diseases, in affected patients would be a great boon to those individuals.